The Origin, Evolution and Conservation of the Arran Sorb Us Microspecies

The Origin, Evolution and Conservation of the Arran Sorb Us Microspecies

THE ORIGIN, EVOLUTION AND CONSERVATION OF THE ARRAN SORB US MICROSPECIES. ASHLEY ROBERTSON A thesis submitted to the University of Edinburgh for the degree of Doctor of Philosophy Institute of Ecology and Resource management, University of Edinburgh July 2000 I I would like to dedicate this thesis to my mother, Yvonne Patricia Robertson. Acknowledgements I would like to thank my supervisors Dr. Adrian Newton, Dr Richard Ennos, Dr Chris Sydes and Dr Juliet Vickery for their time, advice and encouragement. I would especially like to thank Dr Richard Ennos for his advice on the genetic and evolutionary aspects of the project and for his time and patience during the writing of this thesis. Xin- Sheng Hu, John Morman, Amanda Gillies, Anthony Langdon, Nichola, Jenny, Joan, Aileen, Graham French, Sarah Beckesey, Tonny, Sylvia Heredia, Sarah Rendell, Cecile, Rhizana and Graeme Pigott thank you all for your friendship and support. A special thanks to Mick Carr for his friendship, advice on cloning and the odd beer, to Sarah Beckesey and Adele for proofreading, to John Morman for his help and advice throughout, to Nichola Preston for her help and advice with the DNA sequencing, to Tony Church and Philip Lusby for their help, enthusiasm and advice on the Arran whitebeams and to Vicky and Tam for making my trips to Arran very enjoyable and comfortable. To Adele and my family and friends in Somerset a very big thank you. 11 Declaration I declare that this thesis has been composed solely by myself. The work and results reported within are my own, unless otherwise acknowledged. ASHLEY ROBERTSON July 2000, Edinburgh 111 Abstract In theory, populations of agamospermous plants are genetically invariant and consequently hold little evolutionary potential. To test this hypothesis, molecular markers were used to examine evolutionary processes in the Sorbus microspecies S. pseudofennica and S. arranensis, which are endemic to the Isle of Arran, Scotland. The proposed hybrid origin of these microspecies was investigated using a variety of molecular markers. Two isozyme systems (AAT and 6PGD), a nuclear DNA marker and a chloroplast DNA marker all provided markers that were specific to the putative parental species, S. aucuparia and S. rupicola. These markers could not separate S. rupicola from S. aria (a potential parent) or fully distinguish between S. arranensis and S. pseudofennica. Individuals of both S. arranensis and S. pseudofennica were screened and results were compatible with these microspecies being derived from a cross between S. aucuparia and S. rupicola/S. aria. DNA sequences of the chloroplast trnL intron and the trnL-trnF intergenic spacer for the Arran Sorbus microspecies were identical to those of S. aucuparia. In contrast, nucleotide substitutions were evident between the Sorbus microspecies and S. rupicola at three bases. Nuclear DNA markers (five microsatellites and a Rubisco intron primer pair) revealed variation among individuals within S. arranensis and S. pseudofennica microspecies. Only three S. arranensis clones were detected from a total of 179 individuals. In contrast eight clones were detected from a total of 140 S. pseudofennica individuals. Six families of S. arranensis and S. pseudofennica were screened with three nuclear DNA markers. No variation in banding pattern was found among any of the S. arranensis families. In contrast, segregation of molecular makers was found in three out of the six S. pseudofennica families. During the field study, three individual Sorbus trees with a phenotype unlike any previously recorded on Arran were observed at two locations. The gross leaf morphology of these plants appeared to be intermediate between S. aucuparia and S. pseudofennica and resembled that of S. meinichii sensu lato. It was not possible to determine the precise origin of the novel Arran Sorbus from the results of the molecular marker assays. However, their genomes had unique banding patterns and contained markers specific to both S. aucuparia and S. aria sensu lato. The presence of this novel Arran Sorbus hybrid represents previously unrecorded genetic exchanges within the Arran Sorbus complex and these genetic exchanges could possibly lead to the formation of new apomictic taxa. The implications of these results for the understanding of evolutionary processes within agamospermous species, and for the conservation of these threatened taxa are discussed. v Contents Dedication i Acknowledgements Declaration Abstract iv Contents vi List of Tables x List of Figures xi Chapter One Introduction: Evolutionary potential of 1 agamospermous taxa 1.1 Introduction 2 1.2 Evolution 2 1.2.1 Natural selection 4 1.2.2 Sexual reproduction 5 1.2.3 Asexual reproduction 7 1.3 Distribution and origins of agamospermous taxa 9 1.3.1 Distribution of agamospermy 9 1.3.2 Origins of agamospermy 9 1.3.3 Genetic control of agamospermy 11 1.4 Variation within agamospermous taxa 12 1.4.1 Variation within and between populations 12 1.4.2 Genetic structure in asexual Vs sexual populations 13 1.4.3 Origins of clonal diversity 14 1.5 Evolutionary potential of agamosperms 15 1.5.1 Interspecific hybridisation 15 1.5.2 Introgression 15 1.5.3 Homoploid hybrid derivatives 16 1.5.4 Hybrid breakdown 17 1.5.5 Evolutionary potential of agamospermy 18 1.5.6 Evolutionary potential of agamospermous populations 18 1.6 Conclusion 20 1.7 The endemic Sorbus species on Arran 21 1.7.1 Introduction 21 1.7.2 Phylogeny 21 1.7.3 Genetic formulae 22 1.7.4 Breeding systems 22 1.7.5 Variation between species 23 1.8 Objectives 23 vi Chapter Two Testing the proposed hybrid origin of the Arran 26 Sorbus Microspecies 2.1 Introduction 27 2.1.1 Definition of hybridisation 27 2.1.2 Distribution of hybrid taxa 27 2.1.3 Study of hybridisation 27 2.1.4 The use of molecular markers to study plant hybridisation 28 2.1.5 Types of molecular marker 28 2.1.6 Hybndisation within the genus Sorbus 30 2.2 Objectives 31 2.3 Materials and methods 33 2.3.1 Populations of Sorbus arranensis and S. pseudofennica 33 2.3.2 Plant material 34 2.3.2.1 Sorbus microspecies 34 2.3.2.2 Putative parents 34 2.3.3 Electrophoresis procedures 38 2.3.3.1 Method 38 2.3.4 DNA extraction and quantification 39 2.3.4.1 DNA extraction method 39 2.3.4.2 Method for agarose gel electrophoresis 40 2.3.5 DNA amplification 41 2.3.5.1 Rubisco introns 41 2.3.5.2 Chioroplast non-coding regions 42 2.3.6 Alu restriction enzyme digests 43 2.4 Results 44 2.4 1 Isozymes 44 2.4.1.1 Leucine aminopeptidase (LAP) 44 2.4.1.2 Aspartate amino transferase (AAT) 44 2.4.1.3 6- Phosphogluconate dehydrogenase (6PGD) 45 2.4.2 Rubisco intron 49 2.4.3 Chloroplast DNA markers 53 2.4.3.1 Interspecific chloroplast variation 53 2.4.3.2 Maternal parent 53 2.4.3.3 Direction of interspecifc geneflow 53 2.5 Discussion 57 2.5.1 Hybrid origins 57 2.5.2 Chloroplast DNA analysis 62 Chapter Three Genetic structure of Sorbus microspecies, S. 64 arranensis and S. pseudofennica 3.1 Introduction 65 3.2 Objectives 67 3.3 Materials and methods 68 3.3.1 Sample material and DNA extraction 68 3.3.2 Development of nuclear DNA based markers 68 3.3.2.1 PCR-RFLPs 68 vii 3.3.2.2 Cloning and sequencing of anonymous Sorbus DNA 69 3.3.3 DNA amplification 79 3.3.3.1 Rubisco introns 79 3.3.3.2 Microsatellites 79 3.3.4 Data analysis 83 3.4 Results 85 3.4.1 Microsatellite amplification 85 3.4.2 Nuclear DNA primer pair phenotypes 86 3.4.3 S. arranensis and S. pseudoftnnica genotypic variation 92 3.4.3.1 Genotypic variation within S. arranensis and S. pseudofennica 92 3.4.3.2 Genotype diversity within populations 93 3.4.3.3 Genotypic diversity between populations 96 3.5 Discussion 97 3.5.1 Variation between S. arranensis and S. pseudofennica microspecies 97 3.5.2 Genotypic variation within S. arranensis 98 3.5.3 Origin of S. arranensis genotypic diversity 99 3.5.4 Genotypic variation within S. pseudofennica 100 3.5.5 Origin of S. pseudofennica genotypic diversity 101 3.5.6 Comparisons of genotypic diversity 102 Chapter Four Breeding System 107 4.1 Introduction 108 4.2 Objectives 109 4.3 Materials and methods 111 4.3.1 Plantmaterial 111 4.3.2 DNA extraction and quantification 111 4.3.3 DNA amplification 111 4.4 Results 113 4.4.1 Nuclear DNA primer pair phenotypes 114 4.5 Discussion 121 4.5.1 Obligate agamospermy 122 4.5.2 Facultative agamospermy 123 Chapter Five Novel Arran Sorbus Taxon 125 5.1 Introduction 126 5.2 Objectives 127 5.3 Materials and methods 129 5.4 Results 129 5.4.1 Isozyme markers 130 5.4.2 Chioroplast DNA markers 131 5.4.3 Nuclear DNA markers 131 5.5 Discussion 136 5.5.1 Isozyme and Rubisco intron analysis 136 viii Chapter Six Summary and general discussion 138 6.1 Introduction 139 6.2 Hybrid origins 140 6.2.1 Nuclear markers 140 6.2.2 Chloroplast DNA markers 142 6.2.3 Limitations of molecular markers used in hybrid studies 143 6.3 Genetic structure and origins of genotypic diversity 144 6.3.1 Genotypic variation 145 6.3.2 Origin of genotypic diversity 146 6.3.3 Genotypic diversity comparisons between agamospermous and sexual taxa 147 6.3.4 Limitations of molecular markers in studying genotypic diversity 147 6.4 Spatial distribution of S.

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